Hfq links translation repression to stress-induced mutagenesis in E. coli.

Mismatch repair (MMR) is a conserved mechanism exploited by cells to correct DNA replication errors both in growing cells and under nongrowing conditions. Hfq (host factor for RNA bacteriophage Qß replication), a bacterial Lsm family RNA-binding protein, chaperones RNA-RNA interactions between regulatory small RNAs (sRNAs) and target messenger RNAs (mRNAs), leading to alterations of mRNA translation and/or stability. Hfq has been reported to post-transcriptionally repress the DNA MMR gene mutS in stationary phase, possibly limiting MMR to allow increased mutagenesis. Here we report that Hfq deploys dual mechanisms to control mutS expression. First, Hfq binds directly to an (AAN)3 motif within the mutS 5' untranslated region (UTR), repressing translation in the absence of sRNA partners both in vivo and in vitro. Second, Hfq acts in a canonical pathway, promoting base-pairing of ArcZ sRNA with the mutS leader to inhibit translation. Most importantly, using pathway-specific mutS chromosomal alleles that specifically abrogate either regulatory pathway or both, we demonstrate that tight control of MutS levels in stationary phase contributes to stress-induced mutagenesis. By interacting with the mutS leader, Hfq serves as a critical switch that modulates bacteria from high-fidelity DNA replication to stress-induced mutagenesis.

Genome-wide contributions of the MutSα- and MutSß-dependent DNA mismatch repair pathways to the maintenance of genetic stability in Saccharomyces cerevisiae.

The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSα-dependent and MutSß-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level in Saccharomyces cerevisiae. We found that inactivation of MutSα-dependent MMR increases the genome-wide mutation rate by ∼17-fold and loss of MutSß-dependent MMR elevates the genome-wide mutation rate by ∼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSß-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSα-dependent MMR is more important than MutSß-dependent MMR for protection from 1-bp insertions, while MutSß-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5'-NCN-3' trinucleotides, 5'-GCA-3' trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the -1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSß-dependent MMR pathways.

Sensory plastid-associated PsbP DOMAIN-CONTAINING PROTEIN 3 triggers plant growth- and defense-related epigenetic responses.

Sensory plastids are important in plant responses to environmental changes. Previous studies show that MutS HOMOLOG 1 (MSH1) perturbation in sensory plastids induces heritable epigenetic phenotype adjustment. Previously, the PsbP homolog DOMAIN-CONTAINING PROTEIN 3 (PPD3), a protein of unknown function, was postulated to be an interactor with MSH1. This study investigates the relationship of PPD3 with MSH1 and with plant environmental sensing. The ppd3 mutant displays a whole-plant phenotype variably altered in growth rate, flowering time, reactive oxygen species (ROS) modulation and response to salt, with effects on meristem growth. Present in both chloroplasts and sensory plastids, PPD3 colocalized with MSH1 in root tips but not in leaf tissues. The suppression or overexpression of PPD3 affected the plant growth rate and stress tolerance, and led to a heritable, heterogenous 'memory' state with both dwarfed and vigorous growth phenotypes. Gene expression and DNA methylome data sets from PPD3-OX and derived memory states showed enrichment in growth versus defense networks and meristem effects. Our results support a model of sensory plastid influence on nuclear epigenetic behavior and ppd3 as a second trigger, functioning within meristem plastids to recalibrate growth plasticity.

Stochastic organelle genome segregation through Arabidopsis development and reproduction.

Organelle DNA (oDNA) in mitochondria and plastids is vital for plant (and eukaryotic) life. Selection against damaged oDNA is mediated in part by segregation - sorting different oDNA types into different cells in the germline. Plants segregate oDNA very rapidly, with oDNA recombination protein MSH1 a key driver of this segregation, but we have limited knowledge of the dynamics of this segregation within plants and between generations. Here, we reveal how oDNA evolves through Arabidopsis thaliana development and reproduction. We combine stochastic modelling, Bayesian inference, and model selection with new and existing tissue-specific oDNA measurements from heteroplasmic Arabidopsis plant lines through development and between generations. Segregation proceeds gradually but continually during plant development, with a more rapid increase between inflorescence formation and the next generation. When MSH1 is compromised, the majority of observed segregation can be achieved through partitioning at cell divisions. When MSH1 is functional, mtDNA segregation is far more rapid; we show that increased oDNA gene conversion is a plausible mechanism quantitatively explaining this acceleration. These findings reveal the quantitative, time-dependent details of oDNA segregation in Arabidopsis. We also discuss the support for different models of the plant germline provided by these observations.

Molecular dynamics of mismatch detection-How MutS uses indirect readout to find errors in DNA.

The mismatch repair protein MutS safeguards genomic integrity by finding and initiating repair of basepairing errors in DNA. Single-molecule studies show MutS diffusing on DNA, presumably scanning for mispaired/unpaired bases, and crystal structures show a characteristic "mismatch-recognition" complex with DNA enclosed within MutS and kinked at the site of error. But how MutS goes from scanning thousands of Watson-Crick basepairs to recognizing rare mismatches remains unanswered, largely because atomic-resolution data on the search process are lacking. Here, 10 µs all-atom molecular dynamics simulations of Thermus aquaticus MutS bound to homoduplex DNA and T-bulge DNA illuminate the structural dynamics underlying the search mechanism. MutS-DNA interactions constitute a multistep mechanism to check DNA over two helical turns for its 1) shape, through contacts with the sugar-phosphate backbone, 2) conformational flexibility, through bending/unbending engineered by large-scale motions of the clamp domain, and 3) local deformability, through basepair destabilizing contacts. Thus, MutS can localize a potential target by indirect readout due to lower energetic costs of bending mismatched DNA and identify a site that distorts easily due to weaker base stacking and pairing as a mismatch. The MutS signature Phe-X-Glu motif can then lock in the mismatch-recognition complex to initiate repair.

Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA.

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.

Long-read sequencing characterizes mitochondrial and plastid genome variants in Arabidopsis msh1 mutants.

The abundant repeats in plant mitochondrial genomes can cause rapid genome rearrangements and are also a major obstacle in short-read sequencing studies. Nuclear-encoded proteins such as MSH1 are known to suppress the generation of repeat-associated mitochondrial genome variants, but our understanding of these mechanisms has been constrained by the limitations of short-read technologies. Here, we used highly accurate long-read sequencing (PacBio HiFi) to characterize mitochondrial and plastid genome variants in Arabidopsis thaliana msh1 mutant individuals. The HiFi reads provided a global view of recombination dynamics with detailed quantification of parental and crossover recombination products for both large and small repeats. We found that recombination breakpoints were distributed relatively evenly across the length of repeated sequences and detected widespread internal exchanges of sequence variants between pairs of imperfect repeats in the mitochondrial genome of msh1 mutants. Long-read assemblies of mitochondrial genomes from seven other A. thaliana wild-type accessions differed by repeat-mediated structural rearrangements similar to those observed in msh1 mutants, but they were all in a simple low-heteroplasmy state. The Arabidopsis plastid genome generally lacks small repeats and exhibited a very different pattern of variant accumulation in msh1 mutants compared with the mitochondrial genome. Our data illustrate the power of HiFi technology in studying repeat-mediated recombination in plant organellar genomes and improved the sequence resolution for recombinational processes suppressed by MSH1. Plant organellar genomes can undergo rapid rearrangements. Long-read sequencing provides a detailed and quantitative view of mitochondrial and plastid genome variants normally suppressed by MSH1, advancing our understanding of plant organellar genome dynamics.

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes.

Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA, POLIB, MSH1, RECA3, UNG, FPG, and OGG1) for effects on mutation rates in the model angiosperm Arabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations even at low heteroplasmic frequencies. We find that disrupting MSH1 (but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations in msh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within the mutS mismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses. MSH1 has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination.

Social networks in the single cell.

This article comments on: Chustecki JM, Etherington RD, Gibbs DJ, Johnston IG. 2022. Altered collective mitochondrial dynamics in the Arabidopsis msh1 mutant compromising organelle DNA maintenance. Journal of Experimental Botany 73, 5428-5439. Plant mitochondrial DNA (mtDNA) can become damaged in many ways. A major repair mechanism is homologous recombination, which requires an undamaged DNA template. Presumably, this template comes from a different mitochondrion in the same cell. Plant mitochondria undergo fission and fusion to form transient networks which could allow the exchange of genetic information. To test this hypothesis, Chustecki et al. (2022) used msh1 mutants with defective DNA repair, and showed that mitochondrial interactions increased, revealing a link between the physical and genetic behavior of mitochondria.

Altered collective mitochondrial dynamics in the Arabidopsis msh1 mutant compromising organelle DNA maintenance.

Mitochondria form highly dynamic populations in the cells of plants (and almost all eukaryotes). The characteristics and benefits of this collective behaviour, and how it is influenced by nuclear features, remain to be fully elucidated. Here, we use a recently developed quantitative approach to reveal and analyse the physical and collective 'social' dynamics of mitochondria in an Arabidopsis msh1 mutant where the organelle DNA maintenance machinery is compromised. We use a newly created line combining the msh1 mutant with mitochondrially targeted green fluorescent protein (GFP), and characterize mitochondrial dynamics with a combination of single-cell time-lapse microscopy, computational tracking, and network analysis. The collective physical behaviour of msh1 mitochondria is altered from that of the wild type in several ways: mitochondria become less evenly spread, and networks of inter-mitochondrial encounters become more connected, with greater potential efficiency for inter-organelle exchange-reflecting a potential compensatory mechanism for the genetic challenge to the mitochondrial DNA population, supporting more inter-organelle exchange. We find that these changes are similar to those observed in friendly, where mitochondrial dynamics are altered by a physical perturbation, suggesting that this shift to higher connectivity may reflect a general response to mitochondrial challenges, where physical dynamics of mitochondria may be altered to control the genetic structure of the mtDNA population.

Importance of base-pair opening for mismatch recognition.

Mismatch repair is a highly conserved cellular pathway responsible for repairing mismatched dsDNA. Errors are detected by the MutS enzyme, which most likely senses altered mechanical property of damaged dsDNA rather than a specific molecular pattern. While the curved shape of dsDNA in crystallographic MutS/DNA structures suggests the role of DNA bending, the theoretical support is not fully convincing. Here, we present a computational study focused on a base-pair opening into the minor groove, a specific base-pair motion observed upon interaction with MutS. Propensities for the opening were evaluated in terms of two base-pair parameters: Opening and Shear. We tested all possible base pairs in anti/anti, anti/syn and syn/anti orientations and found clear discrimination between mismatches and canonical base-pairs only for the opening into the minor groove. Besides, the discrimination gap was also confirmed in hotspot and coldspot sequences, indicating that the opening could play a more significant role in the mismatch recognition than previously recognized. Our findings can be helpful for a better understanding of sequence-dependent mutability. Further, detailed structural characterization of mismatches can serve for designing anti-cancer drugs targeting mismatched base pairs.

Point mutations in topoisomerase I alter the mutation spectrum in E. coli and impact the emergence of drug resistance genotypes.

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.

The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa.

Over the last 70 years, we've all gotten used to an Escherichia coli-centric view of the microbial world. However, genomics, as well as the development of improved tools for genetic manipulation in other species, is showing us that other bugs do things differently, and that we cannot simply extrapolate from E. coli to everything else. A particularly good example of this is encountered when considering the mechanism(s) involved in DNA mismatch repair by the opportunistic human pathogen, Pseudomonas aeruginosa (PA). This is a particularly relevant phenotype to examine in PA, since defects in the mismatch repair (MMR) machinery often give rise to the property of hypermutability. This, in turn, is linked with the vertical acquisition of important pathoadaptive traits in the organism, such as antimicrobial resistance. But it turns out that PA lacks some key genes associated with MMR in E. coli, and a closer inspection of what is known (or can be inferred) about the MMR enzymology reveals profound differences compared with other, well-characterized organisms. Here, we review these differences and comment on their biological implications.

Bi-allelic variants in DNA mismatch repair proteins MutS Homolog MSH4 and MSH5 cause infertility in both sexes.

STUDY QUESTION: Do bi-allelic variants in the genes encoding the MSH4/MSH5 heterodimer cause male infertility? SUMMARY ANSWER: We detected biallelic, (likely) pathogenic variants in MSH5 (4 men) and MSH4 (3 men) in six azoospermic men, demonstrating that genetic variants in these genes are a relevant cause of male infertility. WHAT IS KNOWN ALREADY: MSH4 and MSH5 form a heterodimer, which is required for prophase of meiosis I. One variant in MSH5 and two variants in MSH4 have been described as causal for premature ovarian insufficiency (POI) in a total of five women, resulting in infertility. Recently, pathogenic variants in MSH4 have been reported in infertile men. So far, no pathogenic variants in MSH5 had been described in males. STUDY DESIGN, SIZE, DURATION: We utilized exome data from 1305 men included in the Male Reproductive Genomics (MERGE) study, including 90 males with meiotic arrest (MeiA). Independently, exome sequencing was performed in a man with MeiA from a large consanguineous family. PARTICIPANTS/MATERIALS, SETTING, METHODS: Assuming an autosomal-recessive mode of inheritance, we screened the exome data for rare, biallelic coding variants in MSH4 and MSH5. If possible, segregation analysis in the patients' families was performed. The functional consequences of identified loss-of-function (LoF) variants in MSH5 were studied using heterologous expression of the MSH5 protein in HEK293T cells. The point of arrest during meiosis was determined by γH2AX staining. MAIN RESULTS AND THE ROLE OF CHANCE: We report for the first time (likely) pathogenic, homozygous variants in MSH5 causing infertility in 2 out of 90 men with MeiA and overall in 4 out of 902 azoospermic men. Additionally, we detected biallelic variants in MSH4 in two men with MeiA and in the sister of one proband with POI. γH2AX staining revealed an arrest in early prophase of meiosis I in individuals with pathogenic MSH4 or MSH5 variants. Heterologous in vitro expression of the detected LoF variants in MSH5 showed that the variant p.(Ala620GlnTer9) resulted in MSH5 protein truncation and the variant p.(Ser26GlnfsTer42) resulted in a complete loss of MSH5. LARGE SCALE DATA: All variants have been submitted to ClinVar (SCV001468891-SCV001468896 and SCV001591030) and can also be accessed in the Male Fertility Gene Atlas (MFGA). LIMITATIONS, REASONS FOR CAUTION: By selecting for variants in MSH4 and MSH5, we were able to determine the cause of infertility in six men and one woman, leaving most of the examined individuals without a causal diagnosis. WIDER IMPLICATIONS OF THE FINDINGS: Our findings have diagnostic value by increasing the number of genes associated with non-obstructive azoospermia with high clinical validity. The analysis of such genes has prognostic consequences for assessing whether men with azoospermia would benefit from a testicular biopsy. We also provide further evidence that MeiA in men and POI in women share the same genetic causes. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation sponsored Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326), and supported by institutional funding of the Research Institute Amsterdam Reproduction and Development and funds from the LucaBella Foundation. The authors declare no conflict of interest.

Bending of Canonical and G/T Mismatched DNAs.

Mismatched base pairs alter the flexibility and intrinsic curvature of DNA. The role of such DNA features is not fully understood in the mismatch repair pathway. MutS/DNA complexes exhibit DNA bending, PHE intercalation, and changes of base-pair parameters near the mismatch. Recently, we have shown that base-pair opening in the absence of MutS can discriminate mismatches from canonical base pairs better than DNA bending. However, DNA bending in the absence of MutS was found to be rather challenging to describe correctly. Here, we present a computational study on the DNA bending of canonical and G/T mismatched DNAs. Five types of geometric parameters covering template-based bending toward the experimental DNA structure, global, and local geometry parameters were employed in biased molecular dynamics in the absence of MutS. None of these parameters showed higher discrimination than the base-pair opening. Only roll could induce a sharply localized bending of DNA as observed in the experimental MutS/DNA structure. Further, we demonstrated that the intercalation of benzene mimicking PHE decreases the energetic cost of DNA bending without any effect on mismatch discrimination.

Sharp kinking of a coiled-coil in MutS allows DNA binding and release.

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.

HDAC6 regulates DNA damage response via deacetylating MLH1.

MutL homolog 1 (MLH1) is a key DNA mismatch repair protein, which plays an important role in maintenance of genomic stability and the DNA damage response. Here, we report that MLH1 is a novel substrate of histone deacetylase 6 (HDAC6). HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLα complex. Moreover, we have identified four novel acetylation sites in MLH1 by MS analysis. The deacetylation mimetic mutant, but not the WT and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSα-MutLα complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSα-MutLα complex by deacetylation of MLH1, leading to the tolerance of DNA damage.

Prevalence of hypermutator isolates of Achromobacter spp. from cystic fibrosis patients.

Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.

The mismatch repair system (mutS and mutL) in Acinetobacter baylyi ADP1.

BACKGROUND: Acinetobacter baylyi ADP1 is an ideal bacterial strain for high-throughput genetic analysis as the bacterium is naturally transformable. Thus, ADP1 can be used to investigate DNA mismatch repair, a mechanism for repairing mismatched bases. We used the mutS deletion mutant (XH439) and mutL deletion mutant (XH440), and constructed a mutS mutL double deletion mutant (XH441) to investigate the role of the mismatch repair system in A. baylyi. RESULTS: We determined the survival rates after UV irradiation and measured the mutation frequencies, rates and spectra of wild-type ADP1 and mutSL mutant via rifampin resistance assay (RifR assay) and experimental evolution. In addition, transformation efficiencies of genomic DNA in ADP1 and its three mutants were determined. Lastly, the relative growth rates of the wild type strain, three constructed deletion mutants, as well as the rifampin resistant mutants obtained from RifR assays, were measured. All three mutants had higher survival rates after UV irradiation than wild type, especially the double deletion mutant. Three mutants showed higher mutation frequencies than ADP1 and favored transition mutations in RifR assay. All three mutants showed increased mutation rates in the experimental evolution. However, only XH439 and XH441 had higher mutation rates than the wild type strain in RifR assay. XH441 showed higher transformation efficiency than XH438 when donor DNA harbored transition mutations. All three mutants showed higher growth rates than wild-type, and these four strains displayed higher growth rates than almost all their rpoB mutants. The growth rate results showed different amino acid mutations in rpoB resulted in different extents of reduction in the fitness of rifampin resistant mutants. However, the fitness cost brought by the same mutation did not vary with strain background. CONCLUSIONS: We demonstrated that inactivation of both mutS and mutL increased the mutation rates and frequencies in A. baylyi, which would contribute to the evolution and acquirement of rifampicin resistance. The mutS deletion is also implicated in increased mutation rates and frequencies, suggesting that MutL may be activated even in the absence of mutS. The correlation between fitness cost and rifampin resistance mutations in A. baylyi is firstly established.

Mechanism of formation of a toroid around DNA by the mismatch sensor protein.

The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS-DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS-DNA complex.